This project will characterize the functional structure of the editosome which catalyzes RNA editing in trypanosomes. It builds on previous studies in the lab that identified editosome genes, determined the functions and that some are essential for editing and editosome structure. It will identify the catalytic, RNA binding and protein interaction domains of editosome proteins, determine their interactions, and their spatial organization in the editosome. The aims are to 1. identify catalytic and RNA binding domains of editosome proteins, 2. characterize interactions among these proteins, 3. identify functional domains in vivo, and, 4. determine the overall editosome structure. Potential functional domains will be identified in silico. Recombinant proteins, fragments thereof, and mutated versions will be assayed for catalysis and RNA binding to identify/localize the functional domains. Standard assays will use various RNA substrates to account for recognition and catalytic specificities. Pair-wise protein interactions and interaction domains will be identified by complementary methods as will multiprotein interactions found in vivo. Functional domains of editosome proteins will be identified and localized and muitiprotein interactions will be characterized in editosomes produced in vivo. Knock-in transgenic trypanosomes will be produced with editosome gene fragments or mutated versions thereof with their expression under regulatory control. They will be tagged for efficient and gentle editosome isolation. The consequences to in vivo editing, editosome structure and function, composition, and association of accessory factors will be determined by various assays, many developed in the lab. The studies will produce a protein/protein/RNA interaction matrix for the editosome. The structure of the editosome and its fragments will be determined by biochemical, electron microscopic, and crystal structure analyses. Overall, the project will determine the three dimensional structure of the editosome and the spatial location of the functional domains within this structure in order to elucidate its functional structure and thus more fully clarify the process of RNA editing.